Molecular determinants of the estrogen receptor-coactivator interface.
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Transcriptional activation by the estrogen receptor is mediated through its interaction with coactivator proteins upon ligand binding. By systematic mutagenesis, we have identified a group of conserved hydrophobic residues in the ligand binding domain that are required for binding the p160 family of coactivators. Together with helix 12 and lysine 366 at the C-terminal end of helix 3, they form a hydrophobic groove that accommodates an LXXLL motif, which is essential for mediating coactivator binding to the receptor. Furthermore, we demonstrated that the high-affinity binding of motif 2, conserved in the p160 family, is due to the presence of three basic residues N terminal to the core LXXLL motif. The recruitment of p160 coactivators to the estrogen receptor is therefore likely to depend not only on the LXXLL motif making hydrophobic interactions with the docking surface on the receptor, but also on adjacent basic residues, which may be involved in the recognition of charged residues on the receptor to allow the initial docking of the motif. (+info)
Estrogen responses in bovine fetal uterine cells involve pathways directed by both estrogen response element and activator protein-1.
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Objectives were to examine possible roles of estrogen receptor (ER) in development of the bovine uterine endometrium in the context of ER type, enhancer type, and ligand-independent activation. Expression vectors producing either ERalpha or ERbeta were introduced into fetal uterine cells from Day 110 to 120 of gestation (UBF120 cells) and into rat embryo fibroblasts (Rat-1 cells), neither of which express endogenous ER. Reporter constructs containing either an estrogen response element (ERE) or activator protein-1 (AP-1) response element were cotransfected. These reporters were also transfected into fetal uterine cells from Day 180 to 200 of gestation (UBF180 cells), which express ER. In UBF120 and Rat-1 cells transfected with either ERalpha or ERbeta, treatment with estradiol-17beta (E2) resulted in increased activity of an ERE reporter construct, but not an AP-1 element reporter construct. The antiestrogen ICI 182,780 (ICI) exhibited E2 antagonist activity with both ERalpha and ERbeta. Thus, all components were present for E2-dependent transcription from an ERE except ER; however, cells were not competent for E2-dependent transcription mediated through AP-1. In UBF180 cells, E2 treatment increased both ERE and AP-1 reporter activity. ICI exhibited E2 antagonist activity. Treatment with epidermal growth factor resulted in increased ERE reporter activity that was inhibited by ICI, indicative of ligand-independent activation of ER. These data suggest that multiple pathways for ER-mediated gene regulation occur in the developing fetal uterus and that nuclear components necessary for action of both ERalpha and ERbeta are present prior to expression of the receptor. (+info)
Vitamin D receptor alleles predict growth and bone density in girls.
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OBJECTIVES: Polymorphism of the vitamin D receptor (VDR), collagen alpha I type I (Col I alpha I), and oestrogen receptor (ER) genes have been shown to account for some of the heritability of bone mineral density (BMD) in adults. This study examined this relation in prepubertal children. METHODS AND SUBJECTS: The relation between genotypes of VDR gene (Taq I, Bsm I, Fok I), Col I alpha I gene (Msc I), and ER gene (Pvu II) with areal BMD, volumetric BMD, and growth were examined in 114 (68 girls) healthy 7 year old, white children. RESULTS: The genotype of the VDR gene (Taq I) correlated with lumbar spine (L1-4) volumetric BMD in girls only, but at no other bone sites. In girls, VDR genotype affected areal BMD at all sites. After adjusting for height and weight, however, this effect was explained completely by the independent effect of the VDR genotype on growth. Girls with genotype TT, were 3.9 kg heavier and 4.1 cm taller than those with tt, but this relation was not present at birth. No relation was found between genotypes of the VDR gene (Fok I), Col I alpha I gene (Msc I), or ER gene (Pvu II) and BMD or growth variables. CONCLUSIONS: In prepubertal girls, VDR alleles contribute to lumbar spine volumetric BMD variance, but the areal BMD effect reflects the relation between areal BMD and growth. VDR alleles might affect postnatal growth regulation. (+info)
Murine Gcm1 gene is expressed in a subset of placental trophoblast cells.
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The gcm gene of Drosophila melanogaster encodes a transcription factor that is an important component in cell fate specification within the nervous system. In the absence of a functional gcm gene, progenitor cells differentiate into neurons, whereas when the gene is ectopically expressed the cells produce excess glial cells at the expense of neuronal differentiation. Recent searches of databases have uncovered high sequence similarity between the Drosophila gem gene and an anonymous human placental cDNA clone (Altschuller et al., 1996; this communication). Here we report the molecular organization of the murine Gcm1, its spatio-temporal pattern of expression in developing placenta, and its map position at E1-E3 on murine chromosome 9. The murine gene is composed of at least 6 exons. The promoter region contains an "initiation sequence" and is GC rich, characteristics of the promoters of several transcription factors. The mRNA has a modest 5'UTR (ca. 200 bases) but an extensive 3' UTR (ca. 2 kb). Northern blot and mRNA in situ hybridization studies showed that Gcm1 expression was readily detectable only in the placenta. It began at embryonic day 7.5 within trophoblast cells of the chorion and continued to about embryonic day 17.5 within a subset of labyrinthine trophoblast cells. Comparison with other transcription factors revealed that Gcm1 expression defines a unique subset of trophoblast cells. (+info)
A mouse mammary tumor virus-Wnt-1 transgene induces mammary gland hyperplasia and tumorigenesis in mice lacking estrogen receptor-alpha.
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Estrogens have important functions in mammary gland development and carcinogenesis. To better define these roles, we have used two previously characterized lines of genetically altered mice: estrogen receptor-alpha (ER alpha) knockout (ERKO) mice, which lack the gene encoding ER alpha, and mouse mammary virus tumor (MMTV)-Wnt-1 transgenic mice (Wnt-1 TG), which develop mammary hyperplasia and neoplasia due to ectopic production of the Wnt-1 secretory glycoprotein. We have crossed these lines to ascertain the effects of ER alpha deficiency on mammary gland development and carcinogenesis in mice expressing the Wnt-1 transgene. Introduction of the Wnt-1 transgene into the ERKO background stimulates proliferation of alveolar-like epithelium, indicating that Wnt-1 protein can promote mitogenesis in the absence of an ER alpha-mediated response. The hyperplastic glandular tissue remains confined to the nipple region, implying that the requirement for ER alpha in ductal expansion is not overcome by ectopic Wnt-1. Tumors were detected in virgin ERKO females expressing the Wnt-1 transgene at an average age (48 weeks) that is twice that seen in virgin Wnt-1 TG mice (24 weeks) competent to produce ER alpha. Prepubertal ovariectomy of Wnt-1 TG mice also extended tumor latency to 42 weeks. However, pregnancy did not appear to accelerate the appearance of tumors in Wnt-1 TG mice, and tumor growth rates were not measurably affected by late ovariectomy. Small hyperplastic mammary glands were observed in Wnt-1 TG males, regardless of ER alpha gene status; the glands were similar in appearance to those found in ERKO/Wnt-1 TG females. Mammary tumors also occurred in Wnt-1 TG males; latency tended to be longer in the heterozygous ER alpha and ERKO males (86 to 100 weeks) than in wild-type ER alpha mice (ca. 75 weeks). We conclude that ectopic expression of the Wnt-1 proto-oncogene can induce mammary hyperplasia and tumorigenesis in the absence of ER alpha in female and male mice. The delayed time of tumor appearance may depend on the number of cells at risk of secondary events in the hyperplastic glands, on the carcinogenesis-promoting effects of ER alpha signaling, or on both. (+info)
Roscovitine induces cell death and morphological changes indicative of apoptosis in MDA-MB-231 breast cancer cells.
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We have previously shown (Mgbonyebi et al., Anticancer Res., 18: 751-756, 1998) that roscovitine, an olomoucine-related purine analogue and a selective inhibitor of cyclin-dependent kinases, inhibited the proliferative activity of human breast epithelial cells in vitro. The purpose of the present study was to identify the cellular processes and targets affected by roscovitine treatment in the estrogen receptor-negative MDA-MB-231 human breast carcinoma cells. Treatment of the cells with 10 microg/ml roscovitine daily for a length of time ranging from 24 to 240 h revealed that the compound inhibited DNA synthesis, induced cell death, and irreversibly inhibited the proliferative activity of the cells. Morphological analysis of roscovitine-treated cells by light and fluorescence microscopy demonstrated that this cyclin-dependent kinase inhibitor induced cell shrinkage, chromatin condensation, reorganization of actin microfilament architecture, and extensive detachment of cells from the cell culture substratum. These cellular events are all known to be associated with apoptosis. Collectively, the data generated from this study suggest that roscovitine induced apoptosis in the estrogen receptor-negative MDA-MB-231 human breast cancer cells. Because the efficacy of many anticancer drugs depends on their ability to induce apoptotic cell death, modulation of this parameter by roscovitine may provide a new chemopreventive and chemotherapeutic strategy for the clinical management of hormone-resistant breast cancers. (+info)
Implication of proteasome in estrogen receptor degradation.
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In MCF-7 breast cancer cells, estradiol (E2) and pure antiestrogen RU 58668 down-regulate the estrogen receptor (ER). Interestingly, the protein synthesis inhibitor cycloheximide (CHX) abrogated solely the effect of E2 suggesting a selective difference in the degradation of the receptor induced by estrogenic and antiestrogenic stimulations. A panel of lysosome inhibitors (i.e. bafilomycin, chloroquine, NH4Cl, and monensin), calpain inhibitors (calpastatin and PD 150606) and proteasome inhibitors (lactacystin and proteasome inhibitor I) were tested to assess this hypothesis. Among all inhibitors tested, lactacystin and proteasome inhibitor I were the sole inhibitors to abrogate the elimination of the receptor induced by both E2 and RU 58668; this selective effect was also recorded in cells prelabeled with [3H]tamoxifen aziridine before exposure to these ligands. Hence, differential sensitivity to CHX seems to be linked to the different mechanisms which target proteins for proteasome-mediated destruction. Moreover, the two tested proteasome inhibitors produced a slight increase of ER concentration in cells not exposed to any ligand, suggesting also the involvement of proteasome in receptor turnover. (+info)
Immunohistochemical detection of estrogen and progesterone receptors in primary breast cancer.
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To evaluate the reliability of the immunohistochemical assay for estrogen receptor (ER) and progesterone receptor (PR) in the prognosis of patients with breast cancer, 83 primary tumors from the patients were studied. Immunohistochemical analysis was performed using antibody ER 1D5 for ER determination and antibody PR-ICA for PR determination. Of all tumors, ER and PR positivities were detected in 36.1% and 45.8% respectively. There was no significant relationship between ER, PR and age of the patients, tumor size or number of involved nodes. However, we found that only the immunohistochemical ER was a predictor of early recurrence in patients with primary breast cancer. In addition, there was no additive effect in recurrence-free survival when both receptor expressions were combined. (+info)